The dynamics of yeast fermentation is of fundamental importance to the success of the brewing process. Hundreds, if not thousands of biochemical reactions transform the wort into the finished beer and through their various interactive combinations and permutations have a cumulative impact on flavor, ethanol content, productivity, colour, aroma, body and foam stability. In the end, these figure largely as determinants of the ultimate quality/cost of the product, and in turn, the consumer demand for it. Over 1,000 compounds have been identified in beer which contribute to its flavor—and each is variously effected by availability of metabolites and processing conditions.
Over the course of typical batch fermentation processes a number of general changes occur. Fermentable sugars are consumed, and unfermentable sugars are converted, and then consumed, all in a more or less orderly sequencing that is related to corresponding ease with which the yeast is able to convert these respective carbohydrate materials into useful energy. The pH of the wort decreases. The yeast cell population goes through a lag phase, then a log phase and finally a stationary phase, before falling into decline.
In addition to the changes listed above, there is a more or less sequential, and generally ordered uptake of amino acids from the wort, (by the yeast), via a complex transport system that is mediated by specific and general permeases. As fermentation progresses, there are large and corresponding fluctuations in the types and concentrations of amino acids that remain usefully available to the yeast population. More specifically, amino acids are classified according to their respective “uptake” characteristics, such as: initial starting time; duration of uptake; and, speed of uptake after inoculation of the medium with yeast (Jones and Pierce, 1964, 1969; Maule et al., 1966; Nakatani et al., 1984 b; Palmqv and Ayrapaa, 1969; Yoshida et al., 1968 a,b). According to this typology, four main groups of amino acids have been defined. Type ‘A’ amino acids are absorbed by yeast from the start of fermentation and are essentially removed from the wort within 20–24 hours. Type ‘B’ amino acids, on the other hand, are taken up after a lag period of 12 hours after which they are rapidly taken up by the yeast. Type ‘C’ amino acids have a longer lag period than type B's before uptake occurs (about 20 hours) and some can even still remain in the final beer if initial wort concentrations are high enough. Type ‘D’ amino acids (e.g. proline), are not assimilated by the yeast in nitrogen rich media. According to the prior art the wort composition has some effects on the rate of uptake of the individual amino acids, but that overall, the uptake of amino acids is generally faithful to this ordered pattern of consumption. This sequential uptake of amino acids is believed to be due to a complex system of amino acid permeases, each having a particular specificity for certain amino acid groups.
The above mentioned fluctuations impact on the enzymatic biosynthesis of valine, particularly during classical type ‘B’ amino acid uptake. This results in cyclical repression/derepression of the metabolic pathways that affect the evolution of extracellular diacetyl. Diacetyl, a vicinal diketone (vicinyl diketone), has a low taste threshold, and imparts a strong buttery/toffee flavor that is considered to be a defect in many beer products.
Diacetyl is, however, a largely incidental by-product of yeast metabolism—and normally serves as an intermediate that is consumed during subsequent fermentation to below perceptual thresholds at some point in time at or near “completion”—or at least after some reasonably brief maturation process. It arises spontaneously (a non-enzymatic oxidation reaction) from precursors in the isoleucine-valine biosynthetic pathway and the need to reduce its concentration to levels below human perceptual thresholds can result in a need for prolonged beer maturation processing, (or other remedial processing), with attendant production costs.
As a result of the aforementioned metabolic dynamics, a first peak in diacetyl concentration normally occurs relatively early in the course of the typical batch fermentation. The diacetyl concentration is subsequently reduced as the yeast takes up diacetyl and converts it enzymatically into acetoin, which in its turn is further metabolized. More specifically, diacetyl is believed to be a by-product of amino acid biosynthesis involving a relationship between amino acid biosynthesis and acetolactate formation, the precursor to diacetyl (Jones and Fink, 1982). Alpha-acetolactate is excreted by the yeast where upon a non-enzymatic oxidative decarboxylation transforms it into diacetyl (Inoue et al., 1968). In parallel to this, in the isoleucine biosynthetic pathway, 2,3-pentanedione is transformed extracellularly from α-aceto-α-hydroxybutyrate, the intermediate, by a similar non-enzymatic decarboxylation reaction. Yeast cells cannot assimilate the extracellular acetohydroxy acids, but they do take up diacetyl and 2,3-pentanedione which undergo enzymatic conversion to acetoin and 3-hydroxy-2-pentanone, respectively. Acetoin is further enzymatically converted to 2,3-butanediol, and 3-hydroxy-2-pentanone to 2,3-pentanediol as seen in FIG. 1. Diacetyl is reduced by the yeast 50 times faster than its rate of formation from α-acetolactate, thus the oxidative decarboxylation step is the rate-determining step (Inoue and Yamamoto, 1970). Diacetyl reduction depends on yeast strain and temperature and decreases gradually in the later stages of fermentation (Haukeli and Lie, 1978).
Problems, however arise when ongoing metabolic fluctuations result in the late fermentation production of diacetyl in amounts that create a second peak in its concentration or otherwise prolongs the presence of an elevated concentration of diacetyl in the wort. The higher diacetyl levels resulting from late fermentation production may not be reduced to below its flavour threshold in a timely manner. The overall concentration of diacetyl in the wort at any given time both during and “post” fermentation, is a combination of the rate of formation of α-acetolactate, its conversion rate, and the rate of reduction of diacetyl to 2,3-butanediol and acetoin. Any latency of elevated diacetyl concentration resulting from late fermentation production thereof, must be reduced before the beer can be further processed—which may necessitate prolonged maturation or other remedial treatments/measures to bring the final diacetyl concentration to within the target specification for the desired end product.
The prior art offers many approaches to the problem of resolving diacetyl issues in beer production.
Amino acids addition has been attempted, in the hope of altering enzymatic regulation of the isoleucine-valine biosynthetic pathway to mitigate diacetyl problems. In general, levels of diacetyl and 2,3-pentanedione throughout the course of fermentation have been found to be related to the initial concentrations of FAN. More specifically, diacetyl and 2,3-pentandione have been effected, in different ways, by different amounts and combinations of the initial concentrations of isoleucine, valine, threonine and leucine in the wort. However, only the addition of valine to wort consistently reduced the amount of diacetyl formed throughout fermentation. No other single or combined supplementation of wort amino acids was shown to have the same effect (Owades et al., 1959; Portno,1966; Maule et al.,1966; Scherrer,1972; Inoue et al.,1973; Nakatani et al., 1984a; Nakatani et al.,1984b). The addition of isoleucine increased diacetyl and or α-acetolactate, but suppressed 2,3-pentanedione (Scherrer (1972; Inoue et al., 1973; Nakatani et al., 1984a; Nakatani et al., 1984b). The addition of threonine gave a small decrease in diacetyl, but increased the amount of 2,3-pentanedione produced (Scherrer, 1972). The relationship between amino acid transport across the membrane and amino acid biosynthesis was demonstrated by Inoue et al. (1973) who showed that there was an inhibition of AHA synthase which reduced the amount of α-acethydroxy acids being produced during the uptake of valine and isoleucine. And on a more practical side, Nakatani et al., 1984a; Nakatani et al., 1984b discuss a minimum FAN level which, if realized, would reduce the maximum amount of vicinyl diketones produced.
Other prior art approaches have focused on wort composition and growth control to reduce diacetyl—based on the showing that high diacetyl may be related to valine exhaustion from the wort before the end of growth (Inoue et al., 1973; Inoue, 1981; Inoue, 1988; Onaka et al., 1985).
Still other prior art offerings include genetic manipulation of the yeast. Efforts to decrease acetolactate synthetase activity were attempted by the Carlsberg group in Denmark (Gjermansen and Sigsgaard, 1987). Much genetic research, making efforts to strengthen the activity of acetolactate reductoisomerase by amplification of the ILV5 region (gene expressing AHA reductoisomerase) has been done (Villanueba, Goossens and Masschelein, 1990; Muthieux and Weiss, 1995; Villa et al., 1995). Work on introducing the acetolactate decarboxylase enzyme from bacteria enables the yeast to convert acetolactate, the precursor of diacetyl, to acetoin directly without affecting other fermentation characteristics (Godtfredsen et al., 1983; Godtfredsen, Lorck and Sigsfaard, 1983; Suihko et al., 1989; Yamano, and Tanaka and Inoue, 1991; Yamano et al., 1995; Takahashi et al. 1995).
The more traditional approach of the brewing industry involves the management of maturation time and/or increased temperature are another method of diacetyl reduction. A heat treatment process during the anaerobic primary fermentation stage can reduce the maturation process as the anaerobic condition allows the conversion of alpha-acetolactate to acetoin directly (Yamauchi et al., 1995a, 1995b; Inoue et al., 1991; Barker and Kirsop, 1973).
Dulieu et al., 1997; 2000, proposed using alpha-acetolactate decarboxylase in an encapsulated fixed bed bioreactor application, to reduce fermentation/maturation time by reducing the amount of alpha-acetolactate that is converted into diacetyl. Notwithstanding the prior art offerings, it would be advantageous to find a way to manage diacetyl formation in beer production that did not require the addition of exogenous enzymes, specialty amino acid addition, genetic manipulation, or complicate processing through energy-expensive heating, or delaying the release of fresh beer in order that protracted holding times would allow the diacetyl to dissipate.